Figure 3

Nhe3 inhibition rescues kidney patterning and ciliopathy phenotypes. (A) Tenapanor treatment reduced the size of the wt1a expression domain in Grk4 knockdown embryos indicating a smaller glomerulus size. n=4 experiments with 218-242 embryos in total. ^****P<0.0001 (data were analyzed using two-way ANOVA with Sidak's multiple comparison post-test). (B) The length of the PST, as detected by trpm7 expression, was reduced in Grk4 morphants by tenapanor. n=3 experiments with 90-100 embryos in total. ^****P<0.0001 (data were analyzed using two-way ANOVA with Sidak's multiple comparison post-test). (C) Tenapanor also decreased the DE length of Grk4 knockdown embryos. n=3 experiments with 74-123 embryos in total. ^****P<0.0001 (data were analyzed using two-way ANOVA with Sidak's multiple comparison post-test). (D) Fluorescence image illustrating the glomerulus and parts of the proximal tubules in Tg(wt1b:GFP) transgenic embryos. Arrows indicate cystic glomeruli, which occur more often in Grk4 MO embryos than in control-injected embryos. Tenapanor reduced the percentage of embryos developing cysts. n=5 experiments with 144-166 embryos in total. ^***P=0.0003 and ^****P<0.0001 (data were analyzed using a two-sided Fisher's exact test with the Bonferroni correction applied). (E) Confocal z-stacks of pronephric tubules stained using anti-PKCz antibody. Grk4 knockdown produces dilated pronephric tubules. Tenapanor reduces the extent of dilatation. n=3 experiments with 25-29 embryos in total. ^****P<0.0001 (data were analyzed using two-way ANOVA with Sidak's multiple comparison post-test). (F) Confocal stacks of cilia visualized by immunofluorescence using an acetylated tubulin (acTub) antibody. Tenapanor also reduced the length of cilia in the distal pronephric tubules in Grk4-depleted embryos. n=3 experiments with 446-639 cilia in total. ^****P<0.0001 (data were analyzed using the Kruskal-Wallis test with Dunn's multiple comparison post-test). (G) Hydrocephalus (arrow) formation in Grk4-depleted embryos was significantly reduced upon treatment with tenapanor. n=5 experiments with 150-167 embryos in total. ^**P=0.0011 and ^****P<0.0001 (data were analyzed using a two-sided Fisher's exact test with the Bonferroni correction applied). Analyses were performed at (A-C) 20 ss and (D-G) 48 hpf. Scale bars: (D and G) 200 μm, and (E and F) 20 μm. Nhe3, sodium-hydrogen exchanger 3; Grk4, G protein-coupled receptor kinase 4; CTRL, control; MO, morpholino oligonucleotide; ss, somite stage; hpf, hours post-fertilization; tenap., tenapanor.